A model for the germ tube formation and mycelial growth form of Candida albicans

A model based on morphological and ultrastructural evidence is presented which illustrates a novel and hitherto undescribed pattern of germ tube formation and hyphal growth in early and mature colonies of Candida albicans. Accordingly, most of the cytoplasm within the parent yeast cell migrates into and forward with the extending germ tubes and leaves behind an extensively vacuolated yeast cell. Growing hyphae similarly are subtended by migrating "slugs' of protoplasm and leave behind vacuolated intercalary compartments. The vacuolated cell compartments apparently must first regenerate their protoplasmic contents before producing branches or secondary germ tubes. This model is used to explain certain unusual features of the growth kinetics of the filamentous form of this organism.     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

A 31P-NMR study of bovine adrenocortical mitochondrial metabolic activities

High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.     

A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].     

Application of in vitro methods to study carbon uptake and transport by AM fungi

Just as multi-compartmented root chambers have advantages over standard plastic pots for the study of nutrient uptake by arbuscular mycorrhizal [AM] fungi in soil, so the split-plate in vitro system has advantages over the standard dual culture system for the study of the physiology of AM fungi. We used the split-plate culture system of Ri T-DNA transformed Daucus carota L. roots and Glomus intraradices Schenck & Smith, in which only the fungus has access to the distal compartment, to study the ability of germ tubes and extraradical and intraradical hyphae to take up ¹³C-labeled substrates. Labeled substrates were added to one side of the plate divider and plates were incubated for 8 weeks while the fungus proliferated on the side from which the root was excluded. Tissues then were recovered from the plate and examined via NMR spectroscopy. Results showed that the morphological phases of the fungus differed in their ability to take up these substrates, most notably that intraradical hyphae take up hexose while extraradical hyphae cannot. In addition, NMR studies indicated that intraradical hyphae actively synthesized lipids while extraradical hyphae did not. These data show that eventual axenic culture of AM fungi is more than a matter of finding the proper substrate for growth. Genetic regulation must be overcome to make extraradical hyphae behave like intraradical hyphae in terms of C uptake and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytoplasmic alkalinization during germ tube formation in Candida albicans

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.     

Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination

Multicelled conidia are formed by many fungal species, but germination of these spores is scarcely studied. Here, the germination and the effects of antimicrobials on multicompartment macroconidia of Fusarium culmorum were investigated. Germ-tube formation was mostly from apical compartments. The intracellular pH (pH(in)) of the different individual cells of the macroconidia was monitored during germination. The pH(in) varied among different compartments and during different stages of germination. The internal pH was lowest in ungerminated cells and rose during germ-tube formation and was highest in new germ tubes. Antifungal compounds affect the pH(in) and differentiation of the conidia. The pH(in) inside the macroconidial compartments was lowered very fast in the presence of nystatin (1 and 4 microg/ml). At sublethal doses (0.3 microg/ml), the apical compartments were preferentially targeted showing lower pH(in) values. The reduced germination capacity of apical compartments under these conditions was compensated by an increased germination capacity of middle compartments.     

In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters

³¹P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from ³¹P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in ³¹P NMR studies.     

Changes in internal and external pH accompanying growth of Candida albicans: studies of non-dimorphic variants

Non-dimorphic variants of Candida albicans, which were unable to form germ tubes or mature hyphae in media containing amino acids, glucose and salts or N-acetylglucosamine or serum, were prepared from two hyphal positive laboratory strains using a physical separation method. The hyphal-minus phenotype was stable and may be due to mutations or phenotypic variation. The variant strains maintained their internal pH within narrower bounds as compared to their parental wild-types. When exposed to conditions that normally supported the induction of germ tubes the cytoplasmic pH of the wild type strains increased from 6.8 to over pH 8.0 within 5 min while in the variants the rise in internal pH was only about 0.3 pH units. The wild type strains acidified the growth medium more rapidly than the variants. The results suggest that the control of internal pH is directly or indirectly associated with the regulation of dimorphism. The variants had unaltered cell volumes and specific growth rates. The hyphal-minus phenotype was however fully reversible since revertants occurred spontaneously on serum containing agar.     

Speculative cell cycle in yeast-phase growth of Sporothrix schenckii: plasma membrane ultrastructure as revealed by freeze-fracture electron microscopy

The cell cycle in yeast-phase growth of Sporothrix schenckii was investigated by light microscopy and freeze-fracture electron microscopy after a 3- to 7-day cultivation on brain heart infusion agar medium at 37 degrees C. Mother yeastlike cells were able to bear daughter yeastlike cells. They were also able to produce germ tubes that had the potential to develop into pseudohyphae and hyphae. On the other hand, hyphae or pseudohyphae born from yeastlike cells were able to bear yeastlike cells directly. These results lead us to propose a hypothetical cell cycle for yeast-phase growth involving yeastlike vegetative cells, pseudohyphae, and hyphae.     

Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans

The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen.     

Intracellular pH in Arbuscular Mycorrhizal Fungi : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H⁺ cotransport of nutrients by arbuscular mycorrhizal hyphae.     

The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization

The location of the septin ring in the germ tubes of Candida albicans hyphae and pseudohyphae was studied using an antibody to Saccharomyces cerevisiae Cdc11p. In pseudohyphae induced by growth at 35 degrees C in YEPD or Lee's medium, a septin ring formed at or near (mean 1.8 microm) the neck between the mother cell and the germ tube. This became double later in the cycle, and the first mitosis took place across the plane of this double ring. A septin ring also formed at the germ tube neck of developing hyphae induced by serum or growth on Lee's medium at 37 degrees C. However, at later times, this ring became disorganized and disappeared. A second double ring then appeared 10-15 microm (mean 12.5 microm) along the length of the germ tube. The nucleus subsequently migrated out of the mother cell into the germ tube, and the first mitosis took place across the plane of this second septin ring. The relocation of the septin ring in developing hyphae provides a clear-cut molecular distinction between hyphae and pseudohyphae. Commitment to one type of septin localization and mitosis was shown to occur early in the first mitotic cycle, well before evagination. Germ tubes of hyphae and pseudohyphae also have different widths. A point of commitment to germ tube width was also demonstrated, but occurred later in the cycle, approximately coincident with the time of evagination.     

Role of nutritional status of the cell in pH regulated dimorphism of Candida albicans

Stationary phase cells of Candida albicans are under the control of glucose repression, as indicated by the inhibition of germ tube formation by glucose. This 'glucose effect' was absent in starved cells which were derived from similar stationary phase cells. Moreover, starved cells required glucose for germ tube formation, suggesting that it was depletion of energy reserves which was the main factor overriding the 'glucose repression machinery' during starvation. High concentration of phosphate in Lee's medium was the reason for the reduced ability of the starved cells to form germ tubes at pH 4.5 (20% of cells compared to 88% at pH 6.8). However, when phosphate was replaced or its concentration reduced, germ tube formation occurred as frequently at pH 4.5 as at pH 6.8. This 'phosphate effect' was not observed in stationary phase cells, as they were already repressed by glucose.     

The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper (Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisome during Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP) and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and mature hyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was not still detectable. When the germlings reached about 40 μm in length, a FM4-64 stained Spk started to be apparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes and mature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFP strain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated only partial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apex of forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Δspa-2 strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate and higher number of branches than the wild type strain, indicating that SPA-2 is required for the stability, behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa, although not for polarity or Spk establishment.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

Ontogeny of the Spitzenkörper in germlings of Neurospora crassa

The Spitzenkörper (Spk) is a highly dynamic and pleomorphic complex located at the hyphal apex of filamentous fungi. Most studies revealing the structure and behavior of the Spk have been conducted on mature vegetative hyphae of filamentous fungi, including both main leading hyphae and branches. However, these reports do not address whether the observations can be extended to germ tubes. By enhanced phase-contrast video-microscopy and laser scanning confocal microscopy we have analyzed the intracellular changes prior to the appearance of a Spk in germlings of Neurospora crassa. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed at the apex of germ tubes. Before a Spk could be observed, young germ tubes (<150 μm) displayed a uniform distribution of organelles such as nuclei, mitochondria, and cytoplasmic granules along the length of the cells. Once the germlings started reaching lengths of more than approximately 150 μm, visible organelles experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles (0.6 ± 0.3 μm) formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk. During this phase the presence of a Spk in the apical dome was not constant. Approximately 30 min later, the immature Spk became more robust and gradually acquired its typical phase-dark appearance, while the growth direction of the germ tube became less wavering. The formation of a mature phase-dark Spk coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.     

落叶松-杨栅锈菌在感病杨树寄主上发育过程的组织学研究

采用荧光染色技术、光学显微镜和电子显微镜技术,系统研究了落叶松-杨栅锈菌在感病杨树叶片上的发育过程。结果表明,在侵染前期（接种12h以内）,锈菌夏孢子在杨树叶片上萌发,利用芽管或附着胞穿透叶表气孔后形成气孔下囊,进而在胞间产生侵染菌丝。进入活体营养生长阶段（接种后24-96h）,锈菌不断产生大量吸器来满足营养需求的同时,侵染菌丝在叶肉细胞间隙蔓延分枝生长至形成菌落结构。最终在产孢阶段（接种120h之后）产孢菌丝分化形成的夏孢子在表皮下聚集成堆,待成熟后突破表皮显露出来。     

Microtubule dynamics and the role of molecular motors in Neurospora crassa

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing beta-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Deltankin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.